nf κb gfp tagged p65  (Addgene inc)

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: Potential of omega-3 and conjugated fatty acids to control microglia inflammatory imbalance elicited by obesogenic nutrients.

doi: 10.1016/j.bbalip.2023.159331

Figure Lengend Snippet: Fig. 5. Omega-3, CLNA and CLA fatty acids can revert Fructose and Palmitic acid-induced microglia inflammatory imbalance elicited by NF-κB pathway activation, ROS production and Src Tyrosine activation. (A) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the miRFP703-Ikβα sensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). A decreased signal means a bigger Ikβα degradation and higher NF-κB pathway activation. Error bar represents the SEM calculated from n > 10 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the GFP-p65 and mneptune biosensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA). The mnpetune probe allows nucleus staining. The results correspond to n of the sensor signal of human microglia expressing the GFP-p65 sensor after the 15 min incubation time. This variation is in relation to the baseline measured for each experiment. A negative signal means a decreased signal in relation to the baseline, meaning that the catalytic p65 subunit of the NF-κB complex migration to the nucleus decreased in cells exposed to a pre-incubation with the studied fatty acids. In cells only subjected to the stimulus solution the signal increased after baseline measure, indicating catalytic p65 subunit translocation to the nucleus. Error bar represents the SEM calculated from n > 10 cells from two independent cultures. One-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the ROS FRET sensor HSP in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). Error bar represents the SEM calculated from n > 15 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Fluorescence imaging and quantification of human microglia cell line (HMC3) expressing the LynSrc FRET sensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). Error bar represents the SEM calculated from n > 15 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre- incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The measurement of the nuclear accumulation of the p65 subunit of NF-κB as a functional indicator of NF-κB activation was achieved by using the NF-κB GFP-tagged p65 (here defined and GFP-p65; addgene plasmid #23255) [82].

Techniques: Activation Assay, Fluorescence, Imaging, Expressing, Incubation, Staining, Migration, Translocation Assay